A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT

Chinopoulos, Christos; Vajda, Szilvia; Csanády, László; Mándi, Miklós; Mathe, Katalin; Ádám-Vizi, Vera

doi:10.1016/j.bpj.2008.12.3915

Cited by 86 publications

(173 citation statements)

References 88 publications

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“…The fluorescent signal (Ex:Em 475:530 nm) was converted to free magnesium concentration by the following equation:

[M g^{2 +}] = K_{d} \frac{F - F_{m i n}}{F_{m a x} - F} - 0.055 m M

where F min and F max represent the signal with saturating concentrations of EDTA or MgCl 2 , respectively. The dissociation constant, K d , is 1mM (Chinopoulos et al, 2009). This measurement was performed following the addition of ATP (5 mM) and sequential PCr additions to a final concentration of 3, 6, 9,12, 15, 18, 21, 24, 30mM.…”

Section: Star ★ Methodsmentioning

confidence: 99%

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

et al. 2018

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SUMMARY Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular “-omics” technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease.

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“…The fluorescent signal (Ex:Em 475:530 nm) was converted to free magnesium concentration by the following equation:

[M g^{2 +}] = K_{d} \frac{F - F_{m i n}}{F_{m a x} - F} - 0.055 m M

Section: Star ★ Methodsmentioning

confidence: 99%

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

et al. 2018

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“…Thus, hydrolysis of MgATP causes an increase in free magnesium ion concentration and subsequent increase in Magnesium Green fluorescence. By invoking binding equilibrium, Magnesium Green has been used in non-imaging studies to determine the ATP-ADP exchange rate through the mitochondrial adenine nucleotide transporter in isolated mitochondria (Chinopoulos et al , 2009) and permeabilized cells (Kawamata et al , 2010). In an imaging study exploiting fluorescence confocal microscopy with isolated hair cells loaded with Magnesium Green, Shin et al .…”

Section: Methods For Detection and Imaging Atpmentioning

confidence: 99%

Imaging Adenosine Triphosphate (ATP)

Rajendran

Dane

Conley

et al. 2016

The Biological Bulletin

100

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Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provides valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific for ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies that are available to visualize ATP in living cells and identify areas where new tools and approaches are needed to expand our capabilities.

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“…6 However, kinetics for their ADP/ ATP exchange activity is largely similar between paralogs, or variable depending on the studies. 7 There has been no clear experimental evidence to date that differential ATP transport kinetics among the paralogs are critical for their specific roles in cells or tissues. Besides ATP transport activity, multiple studies indicate ANT paralogs may differ in their intramitochondrial sublocalization, 8 uncoupling activities, 9 binding partners 10 and effects on mitochondrial permeability transition pore (mPTP) opening and subsequent cellular survival and death control.…”

mentioning

confidence: 99%

Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis

Seo

et al. 2015

Cell Death Differ

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Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress.

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A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT

Cited by 86 publications

References 88 publications

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

Imaging Adenosine Triphosphate (ATP)

Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis

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